Elucidation of the biophysical properties of a protein in solution is a key to understanding its biological activity. Numerous techniques have been developed in the prior art to characterize a protein in solution. They include assays to determine functional activity, immunoreactivity and protein concentration, spectral methods such as Ultra-Violet, Visible, Infra-Red and fluorescence spectroscopy, Circular Dichroism, light scattering, surface plasma resonance, calorimetry, Nuclear Magnetic Resonance, High-Pressure Liquid Chromatography, gel electrophoresis, terminal sequencing analysis, and Mass Spectrometry. Such techniques typically monitor a biophysical property of the protein in solution, such as solubility, activity, ligand binding, aggregation state, and the conformation or folding state.
For example, spectral methods, such as Nuclear Magnetic Resonance (NMR), Circular Dichroism, fluorescence and absorbance spectroscopy provide detailed information regarding the secondary and tertiary structure of proteins in solution, changes in the behavior of a protein under different solvent conditions, comparison of properties between the protein and homologous or mutated forms of the protein, stability of the protein in solution, and structural transitions such as unfolding and refolding under a variety of conditions.
One of the major limitations in studying a biophysical property of a protein by the above techniques is the solubility and stability of the protein in solution. For example, NMR studies of proteins typically require a concentrated protein solution that is stable enough for acquisition of data for several days of more. This requirement has proven to be extremely difficult to satisfy on a consistent basis, and many NMR studies have been delayed or abandoned because of inability to stabilize or solubilize the protein of interest. This factor is likely to become more of a limitation in the future as NMR spectroscopists attempt to study larger proteins.
Typically, a solvent condition for a protein comprises a buffer system that maintains the pH of the solution at or near a constant value and, optionally, contains stabilizers such as salt, detergent, glycerol and excess reductant. The solvent condition in a protein solution is more commonly described as the buffer condition because a solution containing a protein almost always contains a buffer system.
At present, the main approach for identifying buffer conditions for studying the biophysical properties of a protein consists of transferring the protein at relatively low concentration into solutions with various buffer and pH conditions, and then concentrating the solutions and assessing the solubility and stability. Once a buffer and pH have been identified in which the protein is soluble and reasonably stable, an empirical approach is taken to varying stabilizers such as salt, reducing agents, glycerol, detergents, etc., in order to maximize solubility and stability.
In addition to enhancing solubility and stability, a stabilizer should not interfere with the technique employed to study the biophysical property. For example, if a protein solution is to be studied by NMR spectroscopy, then any stabilizer present in the solution should not give rise to resonances that interfere with the NMR spectrum. As another example, if a protein solution is to be studied by ultra-violet absorbance spectroscopy, then any stabilizer present in the protein solution should not strongly absorb ultra-violet radiation in the same frequency range as the protein.
The empirical approach for identifying appropriate buffer conditions is inefficient because the step of transferring the protein into various buffer systems is tedious and time-consuming. Moreover, sampling all of the possible combinations of buffer conditions consumes large amounts of valuable protein. This is a crucial limitation for proteins that are extremely difficult to isolate and purify.
Thus, there is a need for a method to rapidly and efficiently identify a buffer condition in which a protein is soluble and stable. Such a buffer condition would be highly suitable for determining a biophysical property of a protein in solution using any one of the known techniques.
X-ray crystallographers have long had to contend with the converse problem in identifying conditions for precipitating a protein out of solution in order to grow crystals. Currently, the technique of vapor diffusion [1] is used to carry out controlled precipitation, and is combined with incomplete factorial [2] or sparse matrix [3] methods in order to screen large matrices of solvent conditions.
In a typical vapor diffusion method, a protein solution is combined with a precipitating agent and the mixture is sealed within a chamber containing a solvent reservoir in such a way that the solvent is gradually drawn out of the protein solution, leading to supersaturation and precipitation of the protein crystal.
The present invention provides a process for identifying buffer conditions that are suitable for determining a biophysical property of a protein in solution. The process of the present invention uses vapor diffusion, but differs from the crystal growing technique discussed above in that the absence of precipitants and the optional presence of stabilizers allows the optimization of solubility instead of insolubility.